At the Old Rifle uranium mill-tailing site in eastern Colorado, a test of subsurface amendment with acetate to stimulate the reductive immobilization of uranium was monitored by using lipid biomarker analysis and incorporation of 13C-labeled acetate into lipid biomarkers. Both sediment and groundwater samples were analyzed. Within 7 days of acetate addition, groundwater microbial biomass increased by a factor of 5, and remained higher than control values in most samples for the 28 days sampled. At 29 days after the beginning of acetate amendment, 4 of 12 sediment samples had microbial biomass greater than the 95 percent confidence interval of controls. The mole percents of the phospholipid fatty acids 16:1ω7c and 16:1ω5c increased over control values upon acetate amendment, and incorporated high levels of 13C from labeled acetate in groundwater and sediment samples. 16:1ω7c is a biomarker for Geobacter, and evidence is provided that 16:1ω5c represents an unidentified iron-reducing bacterium, probably a member of the Desulfobulbaceae. Biomarkers for organisms other than iron-reducing bacteria, iso- and anteiso-branched fatty acids and 18:1ω9c, decreased upon acetate amendment, and had their highest stable isotope incorporation at least 4 days after labeled acetate amendment ended, evidence for carbon-sharing between iron-reducers and other microorganisms. © 2011 Wiley Periodicals, Inc.